Glucose Protects E. Coli from Death by the Vibrio Cholerae Type VI Secretion System Holly L

Glucose protects E. coli from death by the Vibrio cholerae type VI secretion system Holly L. Nichols1, Cristian Crisan1, Sophia Wiesenfeld1, Gabi Steinbach2, Peter Yunker2, Brian K. Hammer1 1Georgia Institute of Technology, School of Biological Sciences, Atlanta, GA 30332 2Georgia Institute of Technology, School of Physics, Atlanta, GA 30332 Abstract Vibrio cholerae, the causative agent of the intestinal disease cholera, interacts with other bacteria in dense multispecies communities within both host and environmental settings. Using the harpoon-like type VI secretion system (T6SS), V. cholerae delivers toxic effector proteins into neighboring cells, causing cell lysis and death. The T6SS is frequently studied in V. cholerae using a qstR* mutant which constitutively expresses the T6SS. A qstR* V. cholerae strain can effectively kill target species Escherichia coli, Aeromonas veronii., and T6SS-sensistive V. cholerae cells in a standardized lab killing assay, causing a drop in viable cell counts of five orders of magnitude. This study finds that addition of glucose to a standardized killing assay against qstR* V. cholerae restores E. coli survival by three to four orders of magnitude, though the same effect is not found for Aeromonas or T6SS-sensitive V. cholerae. A growth assay revealed that E. coli doubling time does not affect killing by V. cholerae. Additional evidence shows that E. coli does not produce a diffusible molecule that represses the T6SS of V. cholerae. Investigation by fluorescence microscopy revealed that E. coli cells when entirely surrounded by V. cholerae cells survive in the presence but not the absence of glucose, which suggests that glucose causes a relevant physiological change in individual E. coli cells. We propose that further study should focus on the E. coli capsule as a potential mechanism for surviving T6SS attack. This study makes an unprecedented case that attack via the T6SS can be thwarted by sugar metabolism in target cells. Introduction Vibrio cholerae, well known as the bacterium responsible for the diarrheal disease cholera, is also an aquatic microbe. In freshwater and marine environments, V. cholerae can be found in a planktonic state or attached to surfaces of plants, algae, chitinous zooplankton, and even the guts of fish (Takemura et al 2014). Attached to these surfaces in polymicrobial biofilms, V. cholerae can interact, cooperate, and compete with many other bacterial species. One mechanism of bacterial antagonism employed by V. cholerae is the Type VI Secretion System (T6SS), a spike that delivers toxic effector proteins into neighboring cells (Pukatzki et al 2007). When an adjacent bacterial cell is punctured by this contact-dependent mechanism, intoxication from these effectors causes cell lysis. This lethal secretion system is found in 25% of gram-negative bacteria (Bingle et al 2008). The T6SS has been hypothesized to serve many purposes, including biofilm defense, biofilm invasion, elimination of non-kin, and killing of phage-infected bacteria (Russell et al 2014). In addition to the direct effects on bacterial cells, the type VI secretion system in V. cholerae has recently been implicated in improved intestinal colonization in mouse and zebrafish models (Zhao et al 2018, Logan et al 2018). Function of the Type VI Secretion System The activated type VI secretion system is known to deliver a variety of effector proteins that act on different targets within a cell. The suite of effectors found in a particular strain or species is unique, although similarity between effectors in different species suggests effectors can be acquired through horizontal gene transfer (Thomas et al 2017). Cells prevent self-intoxication from these toxic effector proteins by encoding a cognate immunity protein beside each effector in the genome (Dong et al 2013). In the clinical reference strain C6706 of V. cholerae, there are three known effectors with antibacterial properties. TseL is a lipase which breaks down lipids that Diagram 1. Schematic of the Vibrio cholerae type VI secretion comprise the cell membrane (Dong et al system in its extended and contracted state. Contraction of 2013). VasX is a pore-forming protein the T6SS delivers toxic effectors into bacterial target cells, which imbeds in the membrane of the causing cell lysis and death (Crisan unpublished 2018). target cell, and is implicated in antibacterial as well as anti-eukaryotic activity (Dong et al 2013). VgrG-3, the protein which comprises the “tip” of the type VI secretion system harpoon, has a C-terminal peptidoglycan degrading domain (Brooks et al 2013). Together, these three effectors allow C6706 V. cholerae to kill many gram-negative bacterial species including E. coli, Aeromonas veronii, and even V. cholerae cells that are engineered to be deficient in all three cognate immunity proteins. These three species can be used as targets in specialized competition assays called killing assays where V. cholerae and its target are co-cultured on solid media, followed by plating on selective media to enumerate target cells that survived a T6SS attack. Regulation of the Type VI Secretion System The type VI secretion system in V. cholerae clinical strain C6706 is activated by a combination of environmental cues in laboratory settings. When V. cholerae is at high cell density, the transcriptional activator HapR responds by up-regulating T6SS and DNA uptake genes (Borgeaud et al 2015). CytR is involved in sensing nucleoside starvation, and up-regulates the T6SS and DNA uptake under starvation conditions, presumably for the purpose of scavenging nucleosides from target cells lysed by the T6SS (Watve et al 2015, Veening and Blockesh 2017). The regulator TfoX is also involved in this pathway; it senses degraded chitin, which is often liberated by V. cholerae’s chitinases. In clinical strain C6706, induction of all three regulators (HapR, CytR, and TfoX) is required for transcription of qstR, which is another activator of the T6SS (Watve et al 2015, see Diagram 2). While there are additional regulators of the T6SS including TfoY, OscR, and TsrA, overexpression of qstR is sufficient to induce constitutive activity of T6SS in clinical isolates including C6706 (Metzger et al 2017, Ishikawa et al 2012, Zheng et al 2010). V. cholerae mutants with a qstR* mutation are frequently used to study the dynamics of the T6SS in a laboratory environment. Diagram 2. Proposed regulatory scheme for type VI Protection from the Type VI Secretion System secretion in Vibrio cholerae As in many types of biological competition, defense (Watve et al 2015). strategies have evolved in response to the lethal type VI secretion system. The facultative pathogen Pseudomonas aeruginosa has been shown to sense and respond to type VI attack by assembling its own T6SS machinery and firing back at the competitor (Basler et al 2013). Some species have evolved a more guarded strategy. Bacillus fragilis encodes functional immunity proteins that match effectors not encoded in their genome, suggesting a mechanism for resistance against persistent T6SS attack (Wexler et al 2016). This study explores a new mechanism for survival from the T6SS. Previous work has shown that when co-cultured with E. coli in a killing assay, qstR* V. cholerae can reduce the survival of E. coli by 5 orders of magnitude relative to a T6SS- control (Borgeaud et al 2015). However, the current study shows that upon addition of the monosaccharide glucose to a killing assay, the same qstR* V. cholerae only reduced E. coli survival by 1 order of magnitude. This drastic increase in survival is shown to be specific to E. coli target cells and the monosaccharide glucose. E. coli’s survival cannot be explained by differing growth rates or by suppression of the T6SS. Using confocal fluorescence microscopy, E. coli was found to survive in the presence of glucose even when physically surrounded by qstR* V. cholerae cells. This work provides the foundation for further investigation into the mechanism of survival, which is proposed here to be due to increased production of the E. coli capsule in glucose conditions. Methods Type VI Secretion System Killing Assay To measure the strength of the V. cholerae T6SS killing phenotype, a killing assay was adapted from previously described methods (Watve et al 2015). E. coli, V. cholerae, or Aeromonas veronii. cells were grown in overnight at 37˚C in a shaking incubator in liquid Lysogeny Broth (LB) with or without 0.4% glucose. Overnight cultures were diluted to an OD600 of 1.0, and then killer and target cells were mixed in a 10:1 ratio. 50 µL of this mixture was plated on Millipore membrane filters (pore size 0.2 µm) placed on LB agar plates with or without 0.4% glucose. Plates were incubated at 37˚C for 3 hours. After incubation, filters were placed in 50 mL falcon tubes and vortexed in 5 mL LB for 30 seconds. The resulting suspension was serially diluted and plated on selective media to enumerate surviving prey cells, which encoded for resistance to an antibiotic to which the V. cholerae killer was sensitive. Alterations to this standard protocol included changing the target species or the killer strain, using alternative sugars (sucrose, maltose, fructose, lactose, galactose, sucrose), decreasing the concentration of glucose, increasing the killer to target ratio, and including two target species in one killing assay. Luciferase (lux) Assay To measure how the gene expression of the T6SS in V. cholerae varies in glucose, a transcriptional fusion of a major T6SS promoter to the luciferase operon was used. Cells were grown overnight in LB or LB with glucose. These overnight cultures were diluted 1:100 with or without glucose and allowed to grow for 6 hours before luminescence and OD600 measurements were taken to calculate RLU (relative light units). Growth Assays Growth assays on solid LB plates with or without 0.4% glucose were conducted to mimic killing assay conditions.

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